Vertical fluctuations of phospholipid acyl chains in bilayers

The possibility of vertical displacement of acyl chains in lipid bilayers has been examined by quenching the fluorescence of 2-(9-anthroyloxy)palmitic acid with 5- and 16-doxylstearates in dipalmitoylphosphatidylcholine unilamellar vesicles. Measurement of lifetime and steady-state quenching showed that the dynamic component of quenching was independent of the transverse position of the quencher indicating that a quencher at the 16-position could interact with a fluorophore at the 2-position with high frequency. The differences in steady-state quenching could be accounted for by the differences in the static component of quenching. The results provide further evidence for rapid vertical displacements of acyl chains in phospholipid bilayers.     

Gramicidin channels in phospholipid bilayers with unsaturated acyl chains.

In organic solvents gramicidin A (gA) occurs as a mixture of slowly interconverting double-stranded dimers. Membrane-spanning gA channels, in contrast, are almost exclusively single-stranded beta(6,3)-helical dimers. Based on spectroscopic evidence, it has previously been concluded that the conformational preference of gA in phospholipid bilayers varies as a function of the degree of unsaturation of the acyl chains. Double-stranded pi pi(5,6)-helical dimers predominate (over single-stranded beta(6,3)-helical dimers) in lipid bilayer membranes with polyunsaturated acyl chains. We therefore examined the characteristics of channels formed by gA in 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane, 1,2-dioleoylphosphatidylcholine/n-decane, and 1,2-dilinoleoylphosphatidylcholine/n-decane bilayers. We did not observe long-lived channels that could be conducting double-stranded pi pi(5,6)-helical dimers in any of these different membrane environments. We conclude that the single-stranded beta(6,3)-helical dimer is the only conducting species in these bilayers. Somewhat surprisingly, the average channel duration and channel-forming potency of gA are increased in dilinoleoylphosphatidylcholine/n-decane bilayers compared to 1-palmitoyl-2-oleoylphosphatidylcholine/n-decane and dioleoylphosphatidylcholine/n-decane bilayers. To test for specific interactions between the aromatic side chains of gA and the acyl chains of the bilayer, we examined the properties of channels formed by gramicidin analogues in which the four tryptophan residues were replaced with naphthylalanine (gN), tyrosine (gT), and phenylalanine (gM). The results show that all of these analogue channels experience the same relative stabilization when going from dioleoylphosphatidylcholine to dilinoleoylphosphatidylcholine bilayers.     

Quantitative determination of conformational disorder in the acyl chains of phospholipid bilayers by infrared spectroscopy

A method is proposed and demonstrated for the direct determination of conformational disorder (trans-gauche isomerization) as a function of acyl-chain position in phospholipid bilayer membranes. Three specifically deuterated derivatives of dipalmitoylphosphatidylcholine (DPPC), namely 4,4,4',4'-d4-DPPC (4-d4-DPPC), 6,6,6',6'-d4-DPPC (6-d4-DPPC), and 10,10,10',10'-d4-DPPC (10-d4-DPPC), have been synthesized. The CD2 rocking modes in the Fourier transform infrared (FT-IR) spectrum have been monitored as a function of temperature for each derivative. A method originally applied by Snyder and Poore [(1973) Macromolecules 6, 708-715] as a specific probe of hydrocarbon chain conformation in alkanes has been used to analyze the data. The rocking modes appear at 622 cm-1 for a CD2 segment surrounded by a trans C-C-C skeleton and between 645 and 655 cm-1 for segments surrounded by particular gauche conformers. The integrated band intensities of these modes have been used to monitor trans-gauche isomerization in the acyl chains at particular depths in the bilayer. At 48 degrees C, above the gel-liquid-crystal phase transition, the percentage of gauche rotamers present is 20.7 +/- 4.2, 32.3 +/- 2.3, and 19.7 +/- 0.8 for 4-d4-DPPC, 6-d4-DPPC, and 10-d4-DPPC, respectively. The gel phase of the latter two molecules is highly ordered. In contrast, a substantial population of gauche rotamers was observed for the 4-d4-DPPC. The conformational analysis yields a range of 3.6-4.2 gauche rotamers/acyl chain of DPPC above the phase transition. This range is in excellent accord with the dilatometric data of Nagle and Wilkinson [(1978) Biophys. J. 23, 159-175]. The significant advantages of the FT-IR approach are discussed.     

Spontaneous transfer between phospholipid bilayers of dehydroergosterol, a fluorescent cholesterol analog

The spontaneous interbilayer transfer of dehydroergosterol, a fluorescent cholesterol analog, was examined using small unilamellar phospholipid vesicles. The kinetic data were best fit by an equation of the form Aexp (-kt) + B. Qualitatively, the general trend of the half-time for transfer and the base values (B) obtained for dehydroergosterol resemble the corresponding values obtained in the earlier studies of cholesterol transfer. However, quantitative differences, which reflect the molecular structure of the sterol, were observed. Acrylamide quenching performed on the donor vesicles at different stages of the transfer indicated that a time-dependent organization of DHE within the vesicles occurs.     

Partitioning of 1-pyrenesulfonate into zwitterionic and mixed zwitterionic/anionic fluid phospholipid bilayers

Molecular partitioning into biomembranes is of fundamental importance in diverse biochemical processes and reactions. The majority of aqueous/membrane partition data using model membrane systems, is obtained with pure phosphatidylcholine bilayers, being lipid mixtures less used, while studies involving bilayers containing zwitterionic/anionic mixtures of phospholipids are even more scarce. In this study, the solvatochromic effects of 1-pyrenesulfonate observed at 375 nm in aqueous liposome suspensions, and monitored by second derivative absorption spectrophotometry, enabled the determination of its partition constants into defined phospholipid bilayers. We compare, under cautiously settled experimental conditions, the partition of the anionic amphiphile PSA into fluid zwitterionic bilayers of POPC (Kp=6.7 x 10(3), at 25 degrees C), and into fluid mixed zwitterionic/anionic bilayers containing small proportions of anionic phospholipids. At the same temperature, we found increasing K(p) values in parallel with the proportion of POPS mixed with POPC (Kp=3.4 x 10(4) and Kp=7.3 x 10(4), with 5 and 10 mol% of POPS, respectively). Our interpretation is based on the interfacial properties of fluid and flexible mixed zwitterionic/anionic phospholipid bilayers.     

Structure of fully hydrated fluid phase lipid bilayers with monounsaturated chains

Quantitative structures are obtained at 30 degrees C for the fully hydrated fluid phases of palmitoyloleoylphosphatidylcholine (POPC), with a double bond on the sn-2 hydrocarbon chain, and for dierucoylphosphatidylcholine (di22:1PC), with a double bond on each hydrocarbon chain. The form factors F(qz) for both lipids are obtained using a combination of three methods. (1) Volumetric measurements provide F(0). (2) X-ray scattering from extruded unilamellar vesicles provides /F(qz)/ for low q(z). (3) Diffuse X-ray scattering from oriented stacks of bilayers provides /F(qz)/ for high q(z). Also, data using method (2) are added to our recent data for dioleoylphosphatidylcholine (DOPC) using methods (1) and (3); the new DOPC data agree very well with the recent data and with (4) our older data obtained using a liquid crystallographic X-ray method. We used hybrid electron density models to obtain structural results from these form factors. The result for area per lipid (A) for DOPC 72.4 +/- 0.5 A(2) agrees well with our earlier publications, and we find A = 69.3 +/- 0.5 A2 for di22:1PC and A = 68.3 +/- 1.5 A2 for POPC. We obtain the values for five different average thicknesses: hydrophobic, steric, head-head, phosphate-phosphate and Luzzati. Comparison of the results for these three lipids and for our recent dimyristoylphosphatidylcholine (DMPC) determination provides quantitative measures of the effect of unsaturation on bilayer structure. Our results suggest that lipids with one monounsaturated chain have quantitative bilayer structures closer to lipids with two monounsaturated chains than to lipids with two completely saturated chains.     

Lateral pressure dependence of the phospholipid transmembrane diffusion rate in planar-supported lipid bilayers

The dependence of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) flip-flop kinetics on the lateral membrane pressure in a phospholipid bilayer was investigated by sum-frequency vibrational spectroscopy. Planar-supported lipid bilayers were prepared on fused silica supports using the Langmuir-Blodgett/Langmuir-Schaeffer technique, which allows precise control over the lateral surface pressure and packing density of the membrane. The lipid bilayer deposition pressure was varied from 28 to 42 mN/m. The kinetics of lipid flip-flop in these membranes was measured by sum-frequency vibrational spectroscopy at 37°C. An order-of-magnitude difference in the rate constant for lipid translocation (10.9 × 10⁻⁴ s⁻¹ to 1.03 × 10⁻⁴ s⁻¹) was measured for membranes prepared at 28 mN/m and 42 mN/m, respectively. This change in rate results from only a 7.4% change in the packing density of the lipids in the bilayer. From the observed kinetics, the area of activation for native phospholipid flip-flop in a protein-free DPPC planar-supported lipid bilayer was determined to be 73 ± 12 Å²/molecule at 37°C. Significance of the observed activation area and potential future applications of the technique to the study of phospholipid flip-flop are discussed.     

Anion binding to lipid bilayers: a study using fluorescent lipid probes

Anion-induced fluorescence quenching of lipid probes incorporated into the liposomal membrane was used to study the binding of anions to the lipid membrane. Lipid derivatives bearing nonpolar fluorophore located either in the proximity of the polar headgroups (anthrylvinyl-labelled phosphatidylcholine, ApPC; methyl 4-pyrenylbutyrate, MPB) or in the polar region (rhodamine 19 oleyl ester, OR19) of the bilayer were used as probes. The binding of iodide to the bilayers of different compositions was studied. Based on the anion-induced quenching of the fluorescence, the isotherm of adsorption of the quencher (iodide) to the membrane was plotted. For anions, which are non-quenchers or weak quenchers (thiocyanate, perchlorate or trichloroacetate), the binding parameters were obtained from the data of the competitive displacement of iodide by these anions. The association constants of the anion binding to the bilayer (Ka) were determined for the stoichiometry of 1 ion/1 lipid and also for the case of independent anion binding. At the physiological concentration of the salt, which does not bind noticeably to the membrane (150 mM NaCl), anion binding could be satisfactorily described by the Langmuir isotherm. The approach applied here offers new possibilities for the studies of ion-membrane interactions using fluorescent probes.     

Configurations of fatty acyl chains in egg phosphatidylcholine-cholesterol mixed bilayers

Based on the structural properties of cholesterol and egg phosphatidylcholine, and on the assumption that the van der Waals' type attactive interaction between the steroid nucleus and the fatty acyl chains provides a stabilizing force for the cholesterol-egg phosphatidylcholine complex, some specific orientation and configurations of the fatty acyl chains around the steroid nucleus in the interacting system are proposed in terms of an optimal packing. The proposed model suggests that the saturated chains are largely facing the flattened (α) surface of the steroid nucleus of cholesterol, while the unsaturated chains can interact with both the α and β surfaces of the steroid nucleus. It is also suggested that the angular methyl groups on the β surface of the steroid nucleus lock the unsaturated fatty acyl chain in a relatively immobile configuration. Experimental evidence which provides support for the proposed stereochemical model is presented.     

Partitioning of exchangeable fluorescent phospholipids and sphingolipids between different lipid bilayer environments

Exchangeable phospho- and sphingolipid probes (phosphatidylcholine, -ethanolamine, -serine, and -glycerol, phosphatidic acid, sphingomyelin, cerebroside, and sulfatide) have been synthesized in which one acyl chain is substituted with a fluorescent bimanyl, 7-(dimethylamino)coumarin-3-yl, or diphenyl-hexatrienyl group. The distribution of these probes between two different populations of lipid vesicles can be readily monitored by fluorescence intensity measurements, as described by Nichols and Pagano [Nichols, J. W., & Pagano, R. E. (1982) Biochemistry 21, 1720-1726], when one of the vesicle populations contains a low mole fraction of a nonexchangeable quencher, (12-DABS)-18-PC. The probes examined in this study exchange between phospholipid vesicles on a time scale of minutes, with kinetics indicating that the transfer process takes place by diffusion of probe monomers through the aqueous phase. As expected, lipid probes with different charges differ markedly in their equilibrium distributions between neutral and charged lipid vesicles. However, probes with different polar headgroups differ only modestly in their relative affinities for vesicles composed of "hydrogen-bonding" lipids (PE and PS) vs "non-hydrogen-bonding" lipids (PC and PG or O-methyl-PA). Probes with different headgroups also show modest, albeit reproducible, differences in their relative affinities for cholesterol-containing vs cholesterol-free PC/PG vesicles. Our results suggest that lipids with different headgroup structures may mix more nearly ideally in liquid-crystalline lipid bilayers than would be predicted from previous analyses of the phase diagrams for binary lipid mixtures.     

Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers

The fluorescence lifetimes for the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not exhibit single-exponential decays. Rather, the data were fit by biexponential decays with lifetimes of approximately 0.3-0.4 and approximately 0.9-1.3 ns with no significant improvement in chi 2 convergence with the addition of a third component. Average lifetimes were dependent upon lipid phase and to a lesser degree surface charge and the phospholipid head group. In dipalmitoyl-phosphatidylcholine (DPPC)-cholesterol membranes, the C18diI lifetime was sensitive to membrane reorganizations at both 20 and approximately 33 mol % cholesterol. In egg phosphatidylcholine (EPC) bilayers, the C18diI lifetime was essentially independent of its concentration below 1:10(3).     

Interaction between dihydropyridines and phospholipid bilayers: a molecular dynamics simulation

Interaction of the calcium-channel antagonist dihydropyridines (DHPs), lacidipine and nifedipine, with a phospholipid bilayer was studied using 600 ps molecular dynamic simulations. We have constructed a double layer membrane model composed of 42 dimirystoyl-phosphatidylcholine molecules. The DHP molecules locate at about 7 Å from the centre of the membrane, inducing an asymmetry in the bilayer. While lacidipine did not induce significant local perturbations as judged by the gauche-trans isomerisation rate, nifedipine significantly decreased this rate, probably by producing a local rigidity of the membrane in the vicinity of the DHP.     

19F-NMR of fluorine labeled acyl chains in phospholipid bilayer vesicles

¹⁹F-labeled phospholipids, L-α-bis-(ω-fluoro palmitoyl)phosphatidylcholine, L-α-bis-(12,12-difluoro stearoyl)phosphatidylcholine and L-α-bis-(6,6-difluoro palmitoyl)phosphatidylcholine, were incorporated in phospholipid vesicles by sonication of aqueous lipid emulsions. Vesicles were prepared both from the pure fluorine substituted phospholipids as well as from lipid mixtures obtained by combining the fluorine substituted lipids with the synthetic phospholipids, L-α-dilauroyl-, L-α-dimyristoyl-, L-α-dipalmitoyl- and L-α-distearoyl-phosphatidylcholine. Characterization by gel permeation chromatography showed that stable unilamellar vesicles with diameters of ≌200 Å could be obtained with a minimum or absence of multilamellar material. The vesicles give rise to two ¹⁹F resonances in most cases as observed previously [K.J. Longmuir and F.W. Dahlquist, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 2716]. The chemical shift differences undergo systematic changes that confirm the interpretations that the dual ¹⁹F resonances arise from the inner and outer halves of the vesicle bilayer. The shift separation increases systematically as the fluorine label is positioned closer to the phospholipid headgroup and decreases systematically with increasing temperature. Both observations agree with what is currently known about phospholipid vesicle structure. Anomalous results are obtained with DSPC as host vesicle since only a single resonance of inbedded fluorinated phospholipids is found.     

Phenethyl alcohol disorders phospholipid acyl chains and promotes translocation of the mitochondrial precursor protein apocytochrome c across a lipid bilayer.

The interaction of phenethyl alcohol with model membranes and its effect on translocation of the chemically prepared mitochondrial precursor protein apocytochrome c across a lipid bilayer was studied. Phenethyl alcohol efficiently penetrates into monolayers and causes acyl chain disordering judged from deuterium nuclear magnetic resonance measurements with specific acyl chain-deuterated phospholipids. Translocation of apocytochrome c across a phospholipid bilayer was stimulated on addition of phenethyl alcohol indicating that the efficiency of translocation of this precursor protein is enhanced due to a disorder of the acyl chain region of the bilayer.     

Comparison between orientational and conformational orders in fluid lipid bilayers.

The orientational order as determined by 2H NMR and the infrared frequencies of the C--H stretching modes of the methylene groups have been measured for several systems (POPC, POPC/cholesterol and POPE), all in the fluid phase, and then were compared; this work reveals an unexpected linear correlation between them. This experimental result shows that both measurements are essentially sensitive to a common motion, most likely trans/gauche isomerisation. This new correlation with those already found in the literature suggest that several measurements related to the hydrophobic core of the fluid bilayer describe different aspects of a universal behavior. The correlation presented here does not extend to the lipid in gel phase where slower motions affect the NMR lineshape.     

Short range order of hydrocarbon chains in fluid phospholipid bilayers studied by x-ray diffraction from highly oriented membranes

We present a study of the short range ordering of hydrocarbon chains in phospholipid bilayers. The x-ray peak associated with the hydrocarbon chains has been probed by means of reciprocal space mappings. Using 20 keV undulator radiation and samples of negligible mosaicity (orientational disorder), the intensity distribution is probed as a function of two coordinates, the momentum transfer parallel and perpendicular to the bilayer, over a wide range and at high resolution. Structural results are obtained concerning the distribution of tilted segments, the correlation length and the radial distribution function of the quasi two-dimensional liquid structure. A comparison is made with published molecular dynamics data (H. Heller, M. Schaefer, and K. Schulten. 1993. J. Phys. Chem. 97:8343-8360) by direct Fourier transformation of the atomic coordinates. The exact prefactor in the relationship between interchain distance and peak position is derived.     

Effect of the vesicular stomatitis virus matrix protein on the lateral organization of lipid bilayers containing phosphatidylglycerol: use of fluorescent phospholipid analogues

In order to investigate the mode of interaction of peripheral membrane proteins with the lipid bilayer, the basic (pI approximately 9.1) matrix (M) protein of vesicular stomatitis virus was reconstituted with small unilamellar vesicles (SUV) containing phospholipids with acidic head groups. The lateral organization of lipids in such reconstituted membranes was probed by fluorescent phospholipid analogues labeled with pyrene fatty acids. The excimer/monomer (E/M) fluorescence intensity ratios of the intrinsic pyrene phospholipid probes were measured at various temperatures in M protein reconstituted SUV composed of 50 mol % each of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). The M protein showed relatively small effects on the E/M ratio either in the gel or in the liquid-crystalline phase. However, during the gel to liquid-crystalline phase transition, the M protein induced a large increase in the E/M ratio due to phase separation of lipids into a neutral DPPC-rich phase and DPPG domains presumably bound to M protein. Similar phase separation of bilayer lipids was also observed in the M protein reconstituted with mixed lipid vesicles containing one low-melting lipid component (1-palmitoyl-2-oleoylphosphatidylcholine or 1-palmitoyl-2-oleoylphosphatidylglycerol) or a low mole percent of cholesterol. The self-quenching of 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence, as a measure of lipid clustering in the bilayer, was also studied in M protein reconstituted DPPC-DPPG vesicles containing 5 mol % NBD-phosphatidylethanolamine (NBD-PE). The quenching of NBD-PE was enhanced at least 2-fold in M protein reconstituted vesicles at temperatures within or below the phase transition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of macrophages and phospholipid flip-flop in supported lipid bilayers

Subclass-specific antibody-dependent interactions (binding and triggering) between macrophages and supported lipid bilayers have been studied. Percentages of mouse macrophage binding (J774 cell line) to the lipid bilayers were dependent on mouse monoclonal IgG subclasses. The efficiencies were as follows: IgG1 = IgG2a greater than IgG2b greater than IgG3. Furthermore, macrophage triggering (spreading) was more efficient on IgG2a- or IgG1-coated lipid bilayers than on IgG2a, IgG3, or non-specific rabbit IgG. The present experiments show also that phospholipid molecules are able to flip-flop from one side of a supported planar bilayer membrane to the other with a half-life of 10 h-1 day at 25 degrees C.